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INTESTINAL Publication-derived

Intestinal Organoids for Drug Screening and Infection

Full SOP with Academic or a plan

Source Heo et al. · Cincinnati Children's Hospital Medical Center, Cincinnati, USA · 10.1038/s41564-018-0177-8

Authors: Heo I, Dutta D, Schaefer DA, Iakobachvili N, Artegiani B, Sachs N, Boonekamp KE, Bowber G, Huber AR, Riggs MW, Clevers H Duration: 72 days 7 phases Cell source: iPSC-Derived (Human iPSC-Derived Intestinal Organoids)

Abstract

Achieved the first complete Cryptosporidium parvum life cycle in human intestinal organoids through microinjection of oocysts into the organoid lumen. This breakthrough enabled long-term culture of a previously unculturable parasite and established a platform for anti-parasitic drug screening.

Cell source
iPSC-Derived (Human iPSC-Derived Intestinal Organoids)
Application
Parasitic Infection Modeling

Protocol overview

19 steps across 7 phases

Phase 1 · Organoid Culture Preparation Days 0-7
  1. a SI Organoid Culture Setup
  2. b SI Organoid Differentiation
  3. c Lung Organoid Culture Setup
  4. d Prepare Organoids for Microinjection
Phase 2 · Cryptosporidium parvum Preparation Day 0
  1. a Oocyst Preparation for Microinjection
  2. b Sporozoite Preparation for Microinjection
Phase 3 · Microinjection and Infection Day 0
  1. a Prepare Microinjection Apparatus
  2. b Perform Microinjection
  3. c Culture Infected Organoids
Phase 4 · Long-Term Culture (Optional) Days 0-28
  1. a Switching Differentiation Protocol for Parasite Maintenance
Phase 5 · Parasite Detection and Quantification Days 0-6
  1. a RNA Extraction and qRT-PCR
  2. b Immunofluorescence Assay (IFA)
  3. c Electron Microscopy—Scanning Electron Microscopy (SEM)
  4. d Electron Microscopy—Transmission Electron Microscopy (TEM)
Phase 6 · Oocyst Isolation and Viability Testing Days 4-6
  1. a Isolate Oocysts from Organoids
  2. b Mouse Infectivity Assay (Optional but Recommended)
Phase 7 · RNA Sequencing and Transcriptome Analysis Days 0-72
  1. a Sample Preparation for RNA Sequencing
  2. b Library Preparation and Sequencing
  3. c Transcriptome Data Analysis

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Attribution

This SOP was written by Organthis based on the published method in Heo et al.. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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